Product Manager Promega Corporation Madison, WI, United States
Abstract: Cellular responses such as gene expression, enzyme activities, protein synthesis, and protein translocation are orchestrated through activation of diverse cell signaling pathways. In these pathways, phosphorylation of specific proteins by specific kinases constitute important nodes by which the signal is transduced from an upstream activation event to downstream cellular responses. As a corollary, alteration of these kinase activities can have a detrimental effect on these cellular processes, leading to serious pathologies such as cancer, inflammatory diseases, and metabolic diseases. Therefore, monitoring these signaling events through the analysis of specific node phosphorylation states can help us better understand cell behavior in healthy and disease states. To monitor these signaling pathways, we developed the Lumit Cellular Systems kinase immunoassay platform which is a suite of simple homogeneous assays to detect phosphorylation of endogenous proteins of interest in cells. NanoLuc Binary Technology (NanoBiT) is a two-subunit system based on NanoLuc luciferase that was successfully demonstrated for protein-protein interaction (PPI) detection. We generated secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments. A cell lysate containing a target protein is incubated with two primary antibodies (user-supplied) that recognize separate epitopes on the protein. These bring the NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the user-supplied primary antibody pair includes a phospho-specific antibody, the luminescence reflects the level of the target protein phosphorylation. Unlike current assays such as western blot or ELISA that require lengthy sample preparation and multiple wash steps, the Lumit cell-based immunoassay presented here takes less than two hours to complete in a simple homogeneous “Add and Read” format. Moreover, this assay requires only a luminometer for detection. Most importantly, detection of phosphorylated endogenous substrates using this assay is not limited by cell types or transfection efficiency because this assay does not require cell engineering. We tested this system by monitoring the activation of multiple signaling pathways including MAPK signaling, NF-kB, and JAK/STAT, through phosphorylation of specific nodes such as pERK1, pIkB, and pSTAT3. We also tested different small or large molecule inhibitors of these pathways or their corresponding node kinases (MEK1, IKK, or JAKs) and obtained the expected pharmacology. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.